Anti-ASK1 antibody (ab2004)

Thank for the reply and thanks Jennifer's extentisive suggestions. Actually I have already modified the Western cnditions so many times. I will use the cell lines you mentioned to run again and to see what I could get. Actually it's nice when you sell the antibody and you including some positive control cell lysate with it. I bet that would make more custome.

Sincerely,

Haiyan

ANSWER:

Your suggestion to include positive control cell lysates with our products is a good one - thank you for your feedback! We do not currently do this as many of our customers use antibodies for applications other than WB. Instead we have links at the bottom of the datasheet to cell lines in our catalog that can be used as positive controls. You will see there's a link to "SW1353 whole cell lysate, product code ab7915" at the bottom of the online datasheet for ab2004.

I look forward to hearing how you get on in testing the antibody on the recommended lysates.

DEar Dr. Miller,

Actually I already ordered one of your product, ASK1 antibody. I tried several conditions and several celline and primary cell, it did n't work as your data sheet said. Your website said in 60 days, either I could get a refund or replacement, I prefer have a refund.

Sincerely,

Haiyan zhu

ANSWER:

I am sorry to hear that you are still having problems with ab2004. I have traced through your correspondence with Jennifer Priester who has offered extensive suggestions on how you could obtain a positive result with this antibody in your research. Have you now tested this antibody in the positive lysates recommended on the datasheet? (SW1353 whole cell lysate) or perhaps you have tried K562 and A431 cell lysates which Jen also said could be used as positive controls. Please advise and if you are still dissatisfied with this antibody, we will refund you.

Thanks for the replying. Actually, I did 1:500 dilution overnight in several cell line, including human and rat's. Unfortunetelly, I didn't use the positive control as you mentioned because I didn't bought that cell lysate along with ab2004. I just curious, did you test each ab before you put them on market? Also, besides this specific cell lysate, did you test ab2004 in other cell lysate?

ANSWER:

Ab2004 was tested by the antibody's originator before being placed on the market. The antibody was tested in several lysates including SW1353 whole cell lysate and K562 and A431 cell lysates (which can also be used as positive controls).

A few more suggestions - try loading less lysate onto the gel, 20 to 25 ug and try a longer exposure of the membrane to the X-ray film (30 minutes to overnight).

The antibody's originator recommends using the following protocol, which I hope will help you out:

1) Load 20 to 25 microgram of whole cell lysate per lane in an SDS-PAGE mini gel.

2) Run at 20 mA per gel until the dye front is close to the bottom.

3) Transfer the proteins to a nitrocellulose membrane (S&S NCTM) at 250 mA in transfer buffer for 1-4 h, depending on the size of the target protein.

4) Incubate the blot with blocking buffer (5% non-fat dry milk in TBS) overnight at 4°C or 2 hr at room temperature (RT).

5) Incubate the blot with primary antibody (diluted 1:250 to 1:1000 in blocking buffer) for 1 hr in blocking buffer at RT.

6) Wash the blot 3 x 10 min in washing buffer (TBS containing 0.1% Tween 20) with shaking.

7) Incubate blot with anti-rabbit IgG-HRP conjugate (Sigma) (diluted 1:10,000 - 1:20,000 in blocking buffer) for 1 h in blocking buffer at RT.

8) Wash 3 x 10 min in washing buffer with shaking.

9) Drain washing buffer, add ECL solution (Amersham) and develop for 1 min.

10) Expose to X-ray film for 1 to 30 min.

TBS: • 125 mM NaCl • 25 mM Tris pH 8.0 • 0.1% Tween 20

SDS/Running Buffer: • 25 mM Tris • 192 mM Glycine • 0.1% SDS

Transfer Buffer: • 20 mM Tris • 150 mM Glycine • 20% methanol • 0.038% SDS

ORDER NUMBER 33826

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Celll extract

PRIMARY ANTIBODY Abcam antibody 2004

SECONDARY ANTIBODY ZYMED/HRP-conjugated anti-rabbit/1:5000/room tem. 1hr/wqshing in 1XTTBS for 5min,, 15 min, 5 min.

DETECTION METHOD PIERCE/ super signal west pico

ANTIBODY STORAGE CONDITIONS aliquated in -20oC

SAMPLE PREPARATION Buffer with protease inhibitor 4oC

AMOUNT OF PROTEIN LOADED 50 to 75 ug

ELECTROPHORESIS/GEL CONDITIONS Reducing 4-20% or 6%

TRANSFER AND BLOCKING CONDITIONS Buffer with 20%methanol for 80 mins at 90V

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

I'm sorry to hear that you are experiencing trouble with this antibody. I have a few suggestions that will hopefully help you out. First, increase the concentration of the primary antibody. You did not mention what dilutions you tried, but I suggest trying a dilution of 1:500 or higher if needed (please refer to the review on the online datasheet where a customer reported using the antibody at 1:200 and 1:500). Also, incubate the membrane with the primary overnight. Second, make sure that the protein transferred properly to the membrane (try using Ponceaue S). Third, try shorter washes. What type of cell extract are you using? I strongly suggest running a positive control to ensure that the antibody is working properly. On the online datasheet, we recommend SW1353 whole cell lysate (ab7915)as a good positive control for ab2004.

If you have any more questions or continue to experience trouble with this antibody, please contact us again.

i am writing again about the ASK-1 antibody. I just tried the antibody from a differnet company on Jurkat cell lysates without any success.So I need this to work without fail.I am providing as much detail as I can right now and then following up with my questions. AIM 1.To detect endogenous ASK-1 in jurkat cells with the ASK-1 antibody 2. Immune complex kinase assay for ENDOGENOUS ASK-1 activity assay using Mkk6(K/A) for single step assay ( based on Yamamoto,K. et al 1999, Mol Cell biol p8469-8478, Ichijo, H et.al 1997, Science 275:90-94 Nishitoh, H. et .al 1998 cell 2: 389-395) So basically i need the antibody to pull down endogeous ASK-1 to do the kinase assay. My questions: 1 How good / sprcific is the antibody ? 2.How sensitive it is to detect ASK-1 levels ? Can it detect endogenous levels 3.Will this antibody serve the above mentioned experiments? Please go through my refernces to tell me if the ASK-1 anitoboby will work for my system.Then I can order without firther delay.

ANSWER:

Thank you for your enquiry. All the information that we have regarding this antibody is located on the online datasheet. I urge you to refer to the reviews and enquiries tabs on the online datasheet for more information. We recommend using SW1353 whole cell lysate as a positive control and Ab2004 will recognize endogenous ASK 1.